Magnesium is a bivalent metal having the largest content in the living body next to calcium and is an important essential element which is deeply concerned in various enzyme reactions in vivo. The magnesium concentration of serum and plasma changes in competition with the calcium concentration and the changes are observed at the time of central nervous system or heart insufficiency, renal insufficiency, acute pancreatitis and the like, so that the measurement of magnesium is one of the important clinical diagnostics items.
Various methods are known for the colorimetric determination and measurement of magnesium concentration in liquid samples using a bivalent metal chelating indicator. The chelating indicator most frequently used is Xylidyl Blue I. According to Dojin Kagaku Catalog 19th edition (published on May 31, 1994), the calorimetric determination of magnesium can also be effected using Eriochrome Black T, Calcichrome, Carboxyarsenazo, Calmagite, Chlorophosphonazo III, Methyl Thymol Blue, o-Cresolphthalein Complexon, SPANS, Xylenol Orange and the like.
When the calorimetric determination of magnesium in biological samples is carried out using these chelating indicators, it is necessary to add a calcium masking agent in order to avoid the interference of coexisting calcium, independent of the indicators. Examples of the masking agent selective for calcium in the coexistence of magnesium and calcium include O,O'-bis(2-aminomethyl)ethylene glycol-N,N,N',N'-tetraacetic acid (hereinafter often referred to as "GEDTA"), trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (hereinafter often referred to as "CyDTA"), and O,O'-bis(2-aminophenyl)ethylene glycol-N,N,N',N'-tetraacetic acid (hereinafter often referred to as "BAPTA").
Addition of triethanolamine or a cyanide compound is also known as a method for masking other heavy metals such as iron, copper and zinc.
Preparation of a reagent composition for use in the measurement of magnesium by these combinations is now an idea which can easily be deduced by those skilled in the art, and a large number of reagent compositions for use in the measurement of magnesium have been devised and put into practical use. For example, a combination of Xylidyl Blue I with GEDTA is used most commonly, and many reagent kits thereof are now on the market. For example, JP-A-4-120464 discloses a combination of Chlorophosphonazo III with GEDTA or BAPTA (the term "JP-A" as used herein means an "unexamined published Japanese patent application"), EP-A-597900 discloses a combination of Arsenazo I with GEDTA or BAPTA, and JP-A-5-11908 discloses a method in which a formazan derivative is used.
The combination disclosed in U.S. Pat. No. 5,397,710 relates to a dry test piece for total blood use having a blood cell separating function. It uses Eriochrome Black T, Calmagite, Magon, Chlorophosphonazo III, Hydroxynaphthol Blue, Arsenazo I, SPANS or the like as the indicator, and GEDTA as the masking agent.
Of the chelating indicators used in the prior art, every indicator excluding formazan derivatives has an allylazo structure as the chelate action functioning portion and a naphthol structure as the pigment moiety.
Now, when a liquid sample is analyzed using a dry reagent, the sample is mostly un-diluted serum, plasma, urine or the like, and the substance to be measured is contained in a fairly high concentration in many cases. Magnesium is not exceptional, and the concentration in human serum or plasma is normally 1.8 to 2.0 mg/dl and sometimes reaches 5.0 to 6.0 mg/dl in abnormal cases. The test piece must have a capacity to measure such abnormal values.
That is, when a dry test piece for use in the measurement of magnesium is prepared using a metal chelating indicator, the chelating indicator in the reagent must be included in advance in a necessary and sufficient amount for the magnesium concentration in samples (the upper limit concentration of the intended measuring range including abnormal values). However, when the chelating indicator is added in such a necessary and sufficient amount, a significantly high level of coloring is observed even under free and un-developed conditions before forming a complex body with magnesium. In other words, these methods have a disadvantage in that the measuring accuracy of a sample having a low magnesium concentration becomes poor due to the high initial background value.
Since the magnesium concentration in biological fluid, such as serum and plasma, is controlled by considerably strong homeostasis, the normal value is limited to an extremely narrow range. Consequently, since it is important in the diagnosis of diseases to detect a value slightly deviated from the narrow range, the poor measuring accuracy is a fatal disadvantage.
Additionally, when the inventors of the present invention have attempted to prepare various dry test pieces for measuring the magnesium concentration using dry-processing thinkable combinations of the prior art magnesium chelating indicators and calcium masking agents, it was found that the interference of calcium could not be avoided in most of the combinations including the prior art combinations.
Also, many of the thus obtained dry test pieces showed a poor storage stability even under cold storage conditions (4 to 8.degree. C.).